US20090047681A1 - Entropic trapping and sieving of molecules - Google Patents

Abstract

Nanofluidic entropic traps, comprising alternating thin and thick regions, sieve small molecules such as DNA or protein polymers and other molecules. The thick region is comparable or substantially larger than the molecule to be separated, while the thin region is substantially smaller than the size of the molecules to be separated. Due to the molecular size dependence of the entropic trapping effect, separation of molecules may be achieved. In addition, entropic traps are used to collect, trap and control many molecules in the nanofluidic channel. A fabrication method is disclosed to provide an efficient way to make nanofluidic constrictions in any fluidic devices.

Description

• This application claims the benefit of U.S. Provisional Application No. 60/137,146, filed Jun. 1, 1999, the disclosure of which is hereby incorporated herein by reference.
• This invention was made with Government support under Grant no. HG01506, awarded by NIH. The Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
• The present invention relates, in general, to the efficient separation of molecules such as DNA and proteins, and more particularly to a separation device including nanofluidic channels of different sizes for providing alternate thin and thick regions along a channel to act as a filtering or sieving structure.
• The separation of molecules according to their sizes is an essential step in biology and other fields and in analytical procedures such as chromatography, DNA sequencing or genome mapping. Conventional methods for separating molecules include electrophoresis and chromatography, which utilize the different transport properties (mobility) of different molecules in a solution-filled capillary or column. In many cases, additional sieving material, such as a gel matrix, is required to obtain sufficient separation of the molecules to permit analysis. In a conventional gel electrophoresis, as an example, molecules such as DNA molecules are separated during an electric-field-driven motion in a highly restrictive gel matrix, because the mobility of the molecules is dependent on their length. However, this length-dependence of molecule mobility vanishes for DNA molecules longer than about 40,000 base pairs, mainly because the molecules tend to be more stretched and oriented in the direction of the applied electric field. Molecules as long as 10,000,000 base pairs can be separated by pulsing the electric field (pulsed field gel electrophoresis), but this process is usually very time consuming and inefficient.
• To obtain better efficiency and control for separation process, the use of an artificial system using a precisely defined microchannel structure as a molecular sieve has been suggested. However, initial attempts to produce efficient artificial gel systems were hindered by poor understanding of the molecular dynamics in the microchannels. It has been found that the conformation (shape) of DNA or other polymer molecules has a direct impact on their motion in a restrictive medium because the interaction cross section of the molecules with obstacles is changed with conformational change. In free solution, polymer molecules such as DNA have a spherical shape in their equilibrium state, and the size of this equilibrium shape is characterized by a radius of gyration (R_o) of the molecule. In the separation process of DNA or other polymers, it is important to maintain the conformation of the molecule in its equilibrium shape as much as possible, because otherwise the polymeric molecule will stretch out in the direction of the motion, rendering the mobility of the molecule length (size) independent. This is because there is minimal difference in their interaction with a retarding matrix such as gel or obstacles.
• In terms of the fabrication of artificial gel systems, current photolithography techniques are limited in resolution at about the 1 micrometer level. Therefore, one cannot easily make constrictions or obstacles small enough for the separation of important molecules (DNA, proteins etc). Electron beam lithography can fabricate smaller features but it generally is too expensive, and it is difficult to produce a large-area device with this process.
• It became clear that a more careful design of a separation device, combined with an inexpensive technique that can produce many ultrasmall constrictions over a large area, is essential in developing a functioning molecular separation device.
SUMMARY OF THE INVENTION
• When molecules become relaxed or are in their equilibrium spherical shape, their interaction with a retarding matrix can be dependent on the molecule's radius of gyration (R_o), and in turn on the length of the molecule. Accordingly, a design for a molecule sieving structure should include a somewhat open area where molecules can relax, as well as narrow constrictions that can serve as a molecular sieve.
• It is, therefore, an object of the present invention to provide a separation device incorporating nanofluidic constrictions (thin regions) and obstacle free regions (thick regions), through which molecules can be caused to flow either by electrophoresis or by non-electric forces.
• Briefly, the device of the invention provides a flow channel incorporating alternating thin and thick regions which operate as a filter, or sieving structure. The thin regions are sufficiently small to act as constrictions to the flow of small objects, such as DNA molecules, proteins, cells, viruses, or other similarly-sized particles, while the thick regions allow molecules to relax for more efficient separation at the thin region. To this end, the thick region depth may be made comparable to, or substantially larger than, the size of a molecule (for example, the radius of gyration R_ofor polymer molecules) to be sieved. Also the thin region depth may be made substantially smaller than the size of the molecule or other object to be sieved. Although the device of the invention can be used to filter a variety of objects, the following description will be in terms of molecules, and particularly DNA molecules for convenience.
• Accordingly, the present invention is directed to a nanofluidic channel in which the motion of molecules such as DNA molecules is characterized by the provision of molecular traps. In accordance with the invention, an elongated nanofluidic channel is provided with alternating regions of thick and thin gaps along its length. The equilibrium spherical shape of a molecule such as DNA or protein has a radius of gyration R_o, which is the shape the molecule assumes when it is relaxed in an open region, such as in the thick regions of the channel. If the molecule is forced to enter a constriction that is much less than R_o, the molecule has to be deformed from its equilibrium shape. Since such a deformation is entropically unfavorable, a driving force is required to force the molecule to enter the constriction. This effect is referred to as the entropic trapping of a long polymer, and this effect is crucial in the operation of present invention.
• The entropic trapping effect can be utilized in operations such as molecular trapping, molecular band formation, molecular separation and sieving, and molecular flow manipulation in nanofluidic or microfluidic channels. The separation or sieving can be achieved when a suitable driving force is supplied to trapped molecules, when they migrate across many molecular traps and get separated because of the size-dependent trapping effect. Just before the migration through the thin regions, molecules are sieved by entropic trapping effect. After the molecules pass through the thin region, they relax back to their equilibrium shape quickly because of the existence of the thick regions. This process is repeated many times until the required separation is achieved. By controlling the driving force for the molecules, molecular trapping and manipulation can be achieved with the same structure.
• In accordance with one embodiment of the invention, a new method was used to fabricate a nanofluidic channel having narrow constrictions (thin regions), spaced along the length of the channel, with the depth of the thin regions ranging between about 10 nm and about 500 nm, and having relatively thick regions between adjacent constrictions, of between about 0.5 micrometer and about 10 micrometer. Channels of these approximate dimensions may be referred to herein as nanofluidic channels, or simply as nanochannels. In accordance with this method, channels with variable depths were defined and etched in a silicon substrate, or wafer, using two-level photolithography. After a thermal oxidation process, mainly for electrical isolation, the top surface of the device was covered with a thin transparent plate. This technique permitted easy fabrication of very narrow gaps or constrictions without the need for e-beam lithography for the patterning of sub-micrometer features. This process was accomplished by the use of differential etching of two regions and the bonding of a capping layer.
BRIEF DESCRIPTION OF DRAWINGS
• The foregoing and additional objects, features and advantages of the present invention will be apparent to those of skill in the art from the following detailed description of preferred embodiments thereof, taken in conjunction with the accompanying drawings, in which:
• FIG. 1 is a diagrammatic cross sectional view of an entropic trapping nanochannel in accordance with the present invention;
• FIG. 2 is a diagrammatic top plan view of an entropic trap, which illustrates the separation mechanism;
• FIGS. 3 through 7 diagrammatically illustrate the fabrication process for the nanofluidic sieving device of the present invention;
• FIG. 8 illustrates one embodiment of the nanochannel of the invention in combination with cathode and anode electrodes and two loading reservoirs;
• FIG. 9 graphically illustrates the mobility of two different DNA species versus electric field in a nanofluidic sieving channel;
• FIG. 10 illustrates the collection of DNA, launching, separation and detection of DNA bands, in one preferred embodiment of the invention; and
• FIG. 11 is a diagrammatic top plan view of a multiple channel entropic trap device, where two different DNA samples can be loaded and separated simultaneously.
DESCRIPTION OF PREFERRED EMBODIMENT
• Turning now to a more detailed consideration of the present invention, there is illustrated inFIG. 1 ananofluidic sieving device10 in accordance with the present invention. Thedevice10 includes a silicon wafer orsubstrate12 in which is fabricated ananofluidic channel14 having alternatingthick regions16 andthin regions18 along its length. Thechannel14 preferably is covered by atransparent top plate22 which is bonded to thesubstrate12 along the edges of the channel. Thenanochannel14 is filled with a buffer solution or other liquid containing DNA molecules orother polymer molecules20 to be separated. It will be understood that any desired material such as glass or plastic may be used as thesubstrate12, and as thetransparent coverplate22, and any conventional bonding techniques can be used to seal thecoverplate22 to aparticular substrate12.
• In the illustrated embodiment of the invention, which is specific for DNA molecule separation by way of example, thethick regions16 may be between about 0.5 micrometers and 5 micrometers in depth, or thickness, while the thin regions may be between 50 and 200 nm in depth, or thickness. The thicknesses of the thick and thin regions can be varied according to the size of themolecule20 to be separated. Thethin region18 thickness (defined as t_s) is substantially smaller than the radius of gyration R_oof the DNA orother polymer molecule20 to be separated. Thethick region16 thickness (defined as t_d) is compatible to R_oof amolecule20 to be separated, and thus to a typical long DNA or other polymer molecule, allowing the molecule to relax to its equilibrium spherical shape in this region. Because molecules can relax in thethick regions16, they are entropically hindered from entering thethin regions18 of the channel. When amolecule20 to be separated is driven through thenanochannel14 by an electric field or by hydrodynamic pressure, the motion of themolecule20 will be retarded whenever it reaches thethin regions18.
• FIG. 2 is a top view of thenanofluidic sieving channel14 where two different DNA molecules orpolymers20a(smaller) and20b(larger) were driven toward the right-hand end of the channel. Bothmolecules20aand20bare trapped at startingpoints22 of thethin regions18. Thelarger molecule20bhas a wider contact area with thethin region18, as compared with thesmaller molecule20a(w_a<w_b), which makes thelarger molecule20bhave a higher probability of escaping the trapping point and progressing through the channel.
• The length of the thin region18 (defined as l_s) and the length of the thick region16 (defined as l_d) along the length ofelongated nanochannel14 can be varied to accommodate molecules with different R_oand length. Changing l_dchanges the relaxation of the molecule after it escapes thethin region18. As the size of themolecule20 increases, l_dshould be increased to accommodate the increased relaxation time required for big molecules to relax back to equilibrium shape. In the illustrated embodiments, the nanofluidic channel is 30 micrometer wide (W), although other widths can be provided. It will be understood that any desired number of nanochannels with any desired combination of values of l_s, l_d, W, t_s, t_dmay be provided on a wafer, or substrate.
• As illustrated inFIGS. 3 through 7, nanofluidic channels such as thechannels14 illustrated inFIGS. 1 and 2, may be fabricated on asilicon wafer30 by a photolithography and reactive ion etching technique. In an experimental fabrication of nanochannels in accordance with the invention, as illustrated inFIG. 3, achannel32 was defined on thetop surface33 of thewafer30 by standard photolithography, and was etched by a reactive ion etch (RIE), providing a channel having afloor34. Thereafter, as illustrated inFIG. 4, a second level of photolithography and chlorine RIE etching with an oxide mask were used to make spacedthick regions36 within thechannel30. This etching step was performed in thefloor34 of the channel32 (FIG. 3) to produce a second,lower floor37 in each of the thick regions, leaving in the channel a series of paralleltransverse barriers38 spaced apart along the length of thechannel32 between the thick regions. The barriers form the ends of the thick regions of the channel (region16 inFIG. 1) with the tops34 of the barriers forming the thin regions. The structural parameters l_s, l_d, W, t_s, t_dinFIG. 1 can be easily varied during these first two lithography steps with a high precision, and according to the specific needs of the device.
• After completing thechannel32, a pair of loading/unloading apertures40 and42 were fabricated at opposite ends of the channel by potassium hydroxide (KOH) etch-through using a silicon nitride etch mask. One of the twoapertures40 and42 may serve as an inlet for a buffer solution or other liquid, containing molecules to be separated, while the other aperture may serve as the outlet for the solution and the separated molecules. Alternatively, the aperture need not be fabricated, but thechannel32 may instead be connected to other microfluidic or nanofluidic channels or chambers that have different functions, to form an integrated system.
• As illustrated inFIG. 6, a thermal oxide layer50 may be grown on all of the surface of thechannel32 and on the surface of the wafer to a thickness of up to 400 nm to provide electrical isolation between the buffer solution and the silicon substrate. In the case where a non-conducting substrate, orwafer30, such as glass is used, this step may be omitted.
• Finally, as illustrated inFIG. 7, the top of thechannel32 was hermetically sealed with athin glass coverslip52 secured to thetop surface54 of thesilicon substrate30 and its oxide coating50, as by anodic bonding, to provide thenanofluidic channel56. Thecoverslip52 may be a thin Pyrex glass or other suitable material to close the channel and to provide a fluid path across thebarriers38 from theinlet end40 to theoutlet end42. In the case of usingsubstrates30 other than silicon, the hermetic seal may be obtained by suitable bonding techniques such as glass-to-glass fusion bonding or bonding with an intervening thin glue layer. Thecoverplate52 is thin and transparent enough to allow the detection of separated molecules.
• In one preferred embodiment of a nanofluidic channel device, as illustrated inFIG. 8, thenanofluidic sieving device56 illustrated inFIG. 7 is turned upside down, and twoliquid reservoirs58 and60, respectively, are attached.Metallic wires62 and64, preferably noble metals such as platinum or gold, may be inserted into thereservoirs58 and60 respectively, to make acathode66 and an anode68. A voltage V applied across the electrodes produces separation ofmolecules20, which is detected from the bottom side through thetransparent coverplate22 of thedevice10. The detection ofmolecules20, as an example not as a limitation, may be done by using a fluorescent dye attached uniformly to themolecules20 and observing in the channel by anoptical microscope70 or equivalent optical detection system.
• FIG. 9 is a graphical illustration of the mobility of two different (large and small) molecules versus the electric field applied as a driving force to the nanofluidic sieving channel. The driving force for the molecules in the channel may also come from hydrodynamic pressure if desired, and in such a case the pressure will be the relevant quantity, instead of the electric field as given in this example. It is understood that the mobility curves plotted versus the electric field have a sigmoidal shape as shown inFIG. 9. Thecurve80 for larger polymer molecule should be higher than thecurve82 for a smaller polymer in aparticular range84 of the electric field. If electric field is higher (in the range86), the mobility is the same irrespective of the molecule size, because the driving force is too strong and the entropic trapping is negligible. If the electric field is lower (in the range88), then the entropic trapping is so strong that molecules are trapped indefinitely, irrespective of their size. The electric field applied to the nanofluidic channel should be adjusted to the level corresponding to therange84. The specific value for this range may vary for a specific molecules to be separated. If the electric field is adjusted to therange86, all the molecules move at the same speed, irrespective of the size. Therefore, thisrange86 may be used for recollection of already separated molecules or moving the mixture of DNA molecules from one location to another without fractionating them. Theelectric field range88 allows molecules to be collected at the first entropic barrier, because in therange88 the entropic trapping effect is too severe for DNA to overcome even a single entropic barrier within a reasonable amount of time.
• As illustrated inFIG. 10, by way of an example and not limitation, if a number of molecules are supplied to channel14, as by way ofreservoir60 andaperture42, and an electric field in therange88 inFIG. 9 is applied for a specific amount of time along thenanofluidic sieving channel14, one can collect many DNA orpolymer molecules20 at the firstentropic trap90, yielding a highly defined andconcentrated molecule band92. Theconcentrated band92 may be launched into the nanochannel for band separation by switching the electric field from the value in therange88 ofFIG. 9 to the value in therange84 ofFIG. 9. In this illustrated embodiment of the invention, two different types of DNA (20aand20b, small and large DNA, respectively) are mixed in theband92. When launched into the nanochannel, theband92 becomes separated, as it migrates through many entropic traps along the channel, into two bands, afirst band94 and asecond band96. It is understood that the first band is composed oflarger DNA20b, while the second band is composed ofsmaller DNA20a.
• For the detection of this separation, in one preferred embodiment, one may set up a region ofinterest98 and collect the fluorescent signal from thebands94 and96, either optically or using other suitable methods, as a function of time. The separatedbands94 and96, may then be recollected at the other end of the channel sequentially, preferably inaperture40 andreservoir58, or other fluidics channels may be used to redirect each band into separate microfluidic chambers.
• It is imperative to note that the above-mentioned method may be utilized to fractionate mixtures with any number of different types of molecules, as the resolution permits. The resolution may be improved by applying several different optimization techniques. Having a longer channel is one way, but another important method is changing the various structural parameters mentioned inFIG. 1 to get optimized results. For certain polymer molecules, one may optimize a specific set of conditions, including but not limited to, the structural parameters illustrated inFIG. 1, the electric field or theelectric field range84 ofFIG. 10, and the overall length of the nanochannel.
• As diagrammatically illustrated in the top plan view ofFIG. 11, by way of example and not limitation, amultiple channel device98, which is capable of separating multiple samples simultaneously, may be fabricated. In this embodiment of the invention, severalnanofluidic sieving channels100,102,104 and106, each with a different sieving structural parameter, are connected to a larger loading andcollection chamber108. The different structural parameters are optimized for the separation of different length ranges of molecules to be separated. The number of nanochannels which may be connected to a loading orcollection chamber108 may be increased without any difficulty in the fabrication or operation of the device, mainly to accommodate wide variety of molecules. The loading andcollection chamber108 is connected to the cathode by awider channel110, and to a reservoir of sample solution by aloading channel112. In addition, thecentral collection chamber108 is defined by twoentropic barriers114 and116, which enable manipulation of the molecules to be separated, which are in thecentral collection chamber108. Thecentral chamber108, theloading channel112 and thechannel110 are all supported by a supportingpillar structure120, mainly to prevent possible collapse of the coverplate (roof) of the channel down to the bottom.
• In the embodiment of the invention illustrated inFIG. 11, there are twomultiple channel devices98 and98's, having two separate loading andcollection chambers108 connected to two separate sample reservoirs (sample reservoirs A and B). Each loading andcollection chamber108 is connected to the same sets ofnanofluidic sieving channels100,102,104 and106, with various structural parameters, and eventually all of thesenanochannels100 lead to a common anode, whereas the twoloading chambers108 also lead to a common cathode.
• In the operation of the device ofFIG. 11, two different samples of molecules, possibly one unknown sample to be analyzed and one known control or reference sample with size information about the fragments in the sample (in DNA analysis for example, a DNA ladder sample could serve as a reference) may be introduced into sample reservoirs. For loading the molecules into the channels, a suitable electrical potential may be applied between the cathode and the sample reservoirs, causing the molecules to enter theloading channel112, thecentral collection chamber108, and the channel to thecathode110. As a result, thecentral chamber108 would be evenly filled with molecules to be separated. Then another electric field is applied between the cathode and the anode, causing molecule transport to thenanofluidic sieving channels100,102,104 and106. The electric field between the cathode and the anode may be selected to have a value in theelectric field range88 ofFIG. 9, so the molecules are collected at the very first barriers of each nanochannel. With this low electric field, the molecules behind theentropic barrier114 cannot drift into thecentral collection chamber108, but pile up behind thebarrier114. Additionally the existence of thebarrier116 makes sure that the molecules in theloading channel112 do not drift into thecentral chamber108 since there is no substantial electric field existing between the sample reservoir and the cathode. Therefore, only the molecules in thecollection chamber108 can drift into the nanochannels, providing the concentrated band discussed with respect toFIG. 10 which will be launched into each of the nanochannels.
• After this process, the field may again be developed between the cathode and the sample reservoirs, causing the remainder of the molecules behind theentropic barrier114 to be drained back to the sample reservoir, without affecting the collected molecules at the first barriers of thenanochannels100,102,104 and106. This process permits control of the concentration of molecules in the launching band, which is relevant in the separation process. Also, the same process can be repeated as many times as desired, to obtain even higher concentrations of the molecules in the band.
• As the separation process proceeds, the data taken from different samples can be easily detected and compared, enabling more reliable analysis. It is important to know that the number of samples to be analyzed may be increased as desired without any serious technical and operational difficulties.
• Thus, there has been disclosed a nanofluidic channel for use in entropic trapping and sieving of polymer molecules such as DNA and proteins. The channel includes alternating thick and thin segments, or sections, which alternately cause DNA or other polymer molecules to stretch and to return to a rest equilibrium configuration. The channel permits separation of long polymers in a DC applied electric field, with the device structure affecting the mobility of the molecules as they pass through the channels. Entropic traps have other uses in manipulating and collecting many molecules, with a high degree of control, into a narrow band, which is useful in the separation process. Although the invention has been disclosed in terms of preferred embodiments, it will be apparent that variations and modifications may be made without departing from the true spirit and scope thereof as set forth in the following claims.

Claims (16)

1-56. (canceled)

57. A method for analyzing a nucleic acid, comprising:

a) introducing a nucleic acid molecule into an apparatus, wherein the apparatus comprises a chamber having at least a constricted nanofluidic channel connected thereto, wherein the nanofluidic channel is dimensioned to provide an entropic barrier to the nucleic acid;

b) applying a driving force across the nanofluidic channel to force the nucleic acid into the nanofluidic channel in an entropically unfavorable configuration; and

c) analyzing the nucleic acid.

58. A method for analyzing a nucleic acid, comprising:

a) introducing a plurality of nucleic acid molecules of varying length into an apparatus, wherein the apparatus comprises a chamber with an optically-transparent cover plate and a plurality of alternating first unconstricted regions and second constricted nanofluidic channel regions connected thereto, wherein the nanofluidic channel region is dimensioned to provide an entropic barrier to the nucleic acid;

b) applying a predetermined driving force appropriate to drive specific length molecules from the first region entirely into the second region, and to drive molecules longer than the specific length from the first region partially into the second region;

c) reducing or removing the driving force to cause molecules that are longer than the specific length molecules and that are not driven entirely into the second region during the application of the driving force to recoil back into the first region, thus resulting in the separation of molecules based on length, wherein the first region comprises a sufficiently lower free-energy than the second region such that the step of reducing or removing the driving force alone effects the recoil; and

d) analyzing a separated nucleic acid.

59. The method ofclaim 57 or58, wherein the driving force is an electrical potential.

60. The method ofclaim 57 or58, wherein the driving force is hydrodynamic pressure.

61. The method ofclaim 57 or58, wherein the nucleic acid analyzed comprises a label that generates an optically detectable signal.

62. The method ofclaim 61, wherein the analyzing step comprises detecting said label.

63. The method ofclaim 61, wherein the label is fluorescent.

64. The method ofclaim 63, wherein the analyzing step comprises monitoring rate of movement of a fluorescent signal from the nucleic acid molecule analyzed when moving through the nanofluidic channel.

65. The method ofclaim 57, wherein the chamber comprises a plurality of alternating constricted nanofluidic channels and unconstricted regions.

66. The method ofclaim 63, wherein the fluorescent signal detected from the nucleic acid analyzed when located in a constricted nanofluidic channel is compared to a fluorescent signal detected from the nucleic acid analyzed when it is in a unconstricted region.

67. The method ofclaim 57 or58, wherein a varying amount of driving force is applied.

68. The method ofclaim 57 or58, wherein the driving force is applied for a predetermined duration.

69. The method ofclaim 57 or58, wherein the driving force is applied in repeated pulses of fixed duration separated by fixed intervals of reduced or no driving force.

70. The method ofclaim 62, wherein the analyzing step comprises comparing the optically detectable signal from the nucleic acid analyzed with that from a reference nucleic acid of known molecular size or length.

71. The method ofclaim 62, wherein the nucleic acid being analyzed is stretched in said nanofluidic channel.

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